A total of 213 holothurians, representing 16 species, was collected during Project Hourglass, a28-month systematic survey of ten stations along two transects (6-73 m) off central western Florida.This material, supplemented with 8 1 additional Gulf of Mexico specimens supplied by the FloridaDepartment of Natural Resources, brings the total number of species reported in this paper to 20. Ofthese species, 19 have previously been reported from the Gulf of Mexico. One, Allothyone mexicana,can be considered endemic to the Gulf of Mexico, and another, Thyone crassidisca, was recentlydescribed from material including Hourglass specimens. Systematic accounts, pertinent ecologicaldata and line drawings of taxonomically important skeletal elements are included for each species.Keys to all 60 holothurian species known from the Gulf of Mexico are provided. Range extensionsfor several species are noted. (Document contains 79 pages.)